Plant roots affect free-living diazotroph communities in temperate grassland soils despite decades of fertilization

Fixation of atmospheric N2 by free-living diazotrophs accounts for an important proportion of nitrogen naturally introduced to temperate grasslands. The effect of plants or fertilization on the general microbial community has been extensively studied, yet an understanding of the potential combinatorial effects on the community structure and activity of free-living diazotrophs is lacking. In this study we provide a multilevel assessment of the single and interactive effects of different long-term fertilization treatments, plant species and vicinity to roots on the free-living diazotroph community in relation to the general microbial community in grassland soils. We sequenced the dinitrogenase reductase (nifH) and the 16S rRNA genes of bulk soil and root-associated compartments (rhizosphere soil, rhizoplane and root) of two grass species (Arrhenatherum elatius and Anthoxanthum odoratum) and two herb species (Galium album and Plantago lanceolata) growing in Austrian grassland soils treated with different fertilizers (N, P, NPK) since 1960. Overall, fertilization has the strongest effect on the diazotroph and general microbial community structure, however with vicinity to the root, the plant effect increases. Despite the long-term fertilization, plants strongly influence the diazotroph communities emphasizing the complexity of soil microbial communities’ responses to changing nutrient conditions in temperate grasslands.

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Samples with not enough reads were excluded from data analysis, details are reported in the manuscript.
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Blinding was not applicable to this study.
Per fertilization treatment, 6 replicates per plant species (duplicates in each of the three replicate treatment plots) were sampled resulting in 6 bulk soil, 6 rhizosphere, 6 rhizoplane and 6 root samples per investigated plant species.
All plants used in this study were randomly sampled.

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All replication attempts were successful and all biological replicates are displayed in the manuscript.
Samples with not enough reads were excluded from data analysis, details are reported in the manuscript.
Plants and soil from N fertilized, P fertilized and unfertilized treatments were sampled in 2014, NPK fertilized plots were sampled in 2018.
Data were collected during sampling.
Plants were randomly sampled from replicate fertilization treatment sites.
Bulk soil and four plant species with their root-associated compartments were sampled Access to the experimental site was granted from the Agricultural Research Centre in Raumberg-Gumpenstein As little material as possible was sampled for the study Long-term nutrient deficiency experiment, Dystric Cambisol soil, annual temperature:8.5°C,annual precipitation: 1080mm Study the impact of nutrients and plant species on microbial communities in temperate permanent grassland soils.Samples derived from a long-term monitored research site.Per fertilization treatment, 6 replicate plant species (duplicates in each of the three replicate treatment plots) were sampled, resulting in 6 bulk soil, 6 rhizosphere, 6 rhizoplane and 6 root samples per investigated plant species.
All plants used in this study were randomly sampled.
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